Brown adipocyte progenitors in human skeletal muscle and methods for identifying differentiation agents therefor

ABSTRACT

Brown adipose tissue (“BAT”) progenitor cells and methods for identifying BAT progenitor cells in a population of cells are provided. Methods are also provided for inducing differentiation of BAT progenitor cells into differentiated brown adipocytes, inducing expression or increased activity levels of BAT uncoupling protein-1 (“UCP1”), and for identifying agents capable of inducing differentiation of BAT progenitor cells into brown adipocytes and/or inducing expression or increased activity levels of UCP1. Differentiated brown adipocytes and agents and methods for inducing differentiation of BAT progenitor cells can be used for treatment of, or the making of medicaments for the treatment of, metabolic diseases or conditions in a patient such as obesity, overweight, impaired glucose tolerance, insulin-resistance, type 2 diabetes, dyslipidemia, hypertension, cardiovascular diseases, metabolic syndrome, and the like. Differentiated brown adipocytes and agents and methods for inducing differentiation of BAT progenitor cells can be used for prevention of hypothermia.

RELATED APPLICATIONS

This application is a national stage application of InternationalApplication No. PCT/US2009/003217 filed May 27, 2009, which claims thebenefit of and priority to U.S. Provisional Patent Application No.61/071,916, filed May 27, 2008, the contents of both of which are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to brown adipose tissue, progenitor cells, celldifferentiation, and brown adipose tissue uncoupling protein-1. Thedisclosure also relates to metabolic diseases such as obesity, type 2diabetes, insulin-resistance and dyslipidemia.

INTRODUCTION

The epidemic of obesity is closely associated with increases in theprevalence of diabetes, hypertension, coronary heart diseases, cancerand other disorders. The role of white adipose tissue is to storelipids, and it is associated with obesity. The role of the brown adiposetissue (“BAT”) is effectively the opposite. It is specialized in lipidcombustion and the dissipation of energy as heat. Indeed, the brownadipocyte contains lots of mitochondria (in which cellular combustionoccurs) and uniquely expresses BAT uncoupling protein-1 (“UCP1”). UCP1acts as an uncoupler of oxidative phosphorylation, resulting indissipation of energy as heat. The sympathetic nervous system stimulatesmitochondriogenesis and UCP1 expression and activity. BAT-associatedthermogenesis in rodents is increased upon exposure to low temperature(e.g., preventing hypothermia) or as a result of overeating, burningexcess absorbed fat and preventing weight gain. BAT, by modifyingsusceptibility to weight gain and by consuming large amounts of glucose,also improves insulin sensitivity. It therefore plays an important rolein the maintenance of body temperature, energy balance and glucosemetabolism.

Experiments with transgenic animals support the potential anti-obesityproperties of BAT. For example, the genetic ablation of BAT has beenreported to cause obesity, while genetic increase in the amount and/orfunction of BAT (and/or UCP1 expression) reportedly promotes a lean andhealthy phenotype. Specifically, mice with a higher amount of BAT gainless weight and are more insulin-sensitive than control mice. Recently,ectopic BAT depots were evidenced in the mouse muscle, which wereproposed to provide a genetic-based mechanism of protection from weightgain and metabolic syndrome.

Although UCP1 is reported to play a role in the control of energybalance in rodents and UCP1-expressing BAT is present in human neonates,it has long been thought that there was no physiologically relevant UCP1expression in adult humans. Indeed, UCP1-expressing BAT was thought todisappear early in life, and adult humans were thought to be devoid ofBAT.

SUMMARY

Applicants have identified the presence of cells in various tissues thatare capable of differentiating into brown adipocytes. In one aspect,Applicants have identified a population of such cells, which Applicantsrefer to as BAT progenitor cells, in skeletal muscle. The presentdisclosure provides methods for sorting cells from various tissues toidentify and isolate BAT progenitor cells. In some embodiments, BATprogenitor cells are isolated from human skeletal muscle. Methods areprovided for differentiating BAT progenitor cells in vitro and in vivointo brown adipocytes. In some embodiments, BAT progenitor cells can becaused to differentiate in vivo into brown adipocytes in a humansubject.

In some embodiments, BAT progenitor cells of the present disclosure canbe expanded in culture. In another aspect, differentiated BAT progenitorcell UCP1 mRNA expression is increased by agents such as cell-permeatingcAMP derivatives, peroxisome-proliferator-activated receptor (PPARγ)agonists, and the like. BAT progenitor cells that have beendifferentiated into brown adipocytes may, in some embodiments, containlarge amounts of mitochondrial transcription factor A (mtTFA) and PPARγcoactivator-1α (PGC-1α), which are both involved in the control ofmitochondriogenesis, as well as of mitochondrial marker cytochromeoxidase IV (COX IV). Differentiated BAT progenitor cells can exhibit oneor more of the following characteristics: high levels of UCP1expression, high levels of uncoupled respiration, high metabolic rate.Applicants provide differentiated cells that are equipped to metabolizeglucose, oxidize fatty acids, and dissipate energy as heat viauncoupling of oxidative phosphorylation.

The present disclosure provides methods for detection of UCP1 mRNA inthe skeletal muscle of adult humans, and methods for increasing itsexpression in vivo. Although prior studies concerning UCP1 expression inadult humans have focused on white adipose tissue, applicants disclosethe existence in, and isolation from, human skeletal muscle of brownadipose progenitor cells with a substantial potential for UCP1expression. In some embodiments, this reservoir of BAT progenitor cellscan be utilized for modulation of energy dissipation and for treatingobesity, diabetes, and metabolic diseases.

In some aspects, this disclosure provides methods for the identificationof BAT progenitor cells in human skeletal muscle and methods to isolatethese cells from human skeletal muscle samples. Also provided areconditions and agents (e.g., compounds, proteins, biologicals, and thelike) that promote the differentiation of these progenitor cells tobrown adipocytes in vitro, in vivo, or both. Methods are provided forusing these conditions and agents to treat metabolic diseases such asobesity, type 2 diabetes, insulin-resistance, dyslipidemia, and thelike.

The present disclosure provides assays that allow identification ofagents (e.g., compounds, proteins, biologicals, and the like) thatinduce the expression of the UCP1 gene, promote the differentiation ofBAT progenitor cells into brown adipocytes in vitro, promote thedifferentiation of BAT progenitor cells to brown adipocytes in vivo, orcombinations of these activities. According to some embodiments, agentsidentified in this manner can be used to treat metabolic diseases suchas obesity, type 2 diabetes, insulin-resistance, dyslipidemia, and thelike.

These and other features of the present disclosure are set forth herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show immunohistochemical description and FACS analysis andsorting of vascular cells in human fetal muscle. FIG. 1(A) scale bar is50 μm.

FIGS. 2A-E show culture under adipogenic conditions of cells sorted fromhuman fetal muscle and RT-PCR and Western blot analysis for the CD34+cells. FIGS. 2(A), (B), (C): Phase contrast; scale bar: 50 μm.

FIGS. 3A-C show uncoupling of mitochondrial respiration and control ofUCP1 mRNA expression in human fetal muscle CD34+ cells.

FIGS. 4A-C show characterization of adult muscle and WAT cells inadipogenic culture. FIGS. 4(A), (C): Phase contrast; scale bar: 50 μm.

FIG. 5 shows effects of rosiglitazone on UCP1 mRNA expression in humanskeletal muscle.

DESCRIPTION OF VARIOUS EMBODIMENTS

The present disclosure provides methods for identifying and isolatingBAT progenitor cells in and from various tissues, including, in someembodiments, the identification of common brown adipocyte progenitorcells in human skeletal muscle and isolation of such cells from humanskeletal muscle samples. In some embodiments, the cell sorting can bedone by immunohistochemical analysis of cell surface markers such ascluster of differentiation/designation (“CD”) molecules CD34, CD45,CD56, and CD146. Hematopoetic cells and myogenic progenitors can besorted based on identification of CD45 and CD56, respectively, on theircell surfaces. CD34 and CD146 can be used to identify endothelial cellsand pericytes, respectively. In one aspect, expression of CD34identifies a cell as a progenitor of a brown adipocyte.

Flow cytometry, fluorescent-activated cell sorting (“FACS”), and othercell sorting techniques known in the art can be used for sorting cellsobtained from various tissues and for separating BAT progenitor cellsfrom other cells. Among other techniques known in the art, multi-colorFACS can be used to identify CD34+ endothelial cells and CD146+pericytes and separate them from each other and from CD45+ hematopoieticcells and CD56+ myogenic progenitors. Reverse transcriptase polymerasechain reaction (“RT-PCR”) analysis can be used to confirm the absence ofhematopoietic cells and myogenic progenitors from the populations ofCD34+ and CD146+ cells.

Applicants have found that a population of progenitors is present inskeletal muscle, and that this population is, in some embodiments, foundin skeletal muscle but not in white adipose tissue and, in someembodiments, exclusively found in skeletal muscle (i.e., not in othertissues). The skeletal muscle may be that of a human or of any animal,and populations of progenitor cells may be diffuse in the skeletalmuscle or concentrated in discrete regions. BAT progenitor cells may, insome embodiments, be found between myofibers. Skeletal muscle BATprogenitor cells may be a stationary population or may be mobile bothwithin skeletal muscle or other tissue and between and among differenttissues. Further, BAT progenitor cells can be found in fetal, juvenile,and adult skeletal muscle.

The present teachings provide BAT progenitor cells isolated from varioustissues. For example, BAT progenitor cells isolated from human skeletalmuscle are provided. In some embodiments, the BAT progenitor cells arefound in skeletal muscle but not in white adipose tissue, and/or areexclusively found in skeletal muscle. Some BAT progenitor cells mayexpress UCP1, mitochondrial transcription factor A (mtTFA), and/or PPARγcoactivator-1α (PGC-1α) as well as one or more of the correspondingmRNAs. The present disclosure provides methods for detection of BATprogenitor cells and/or UCP1 mRNA in the skeletal muscle of adulthumans. Although prior studies concerning UCP1 expression in adulthumans have focused on white adipose tissue, applicants disclose theexistence in, and isolation from, human skeletal muscle of brown adiposeprogenitor cells with a high potential for UCP1 expression. In someembodiments, a reservoir of BAT progenitor cells in skeletal muscleprovides a mechanism for modulating energy dissipation for treatment ofmetabolic diseases such as obesity, diabetes, and the like.

At least a portion of the population of progenitor cells present inskeletal muscle is capable of differentiating into genuine brownadipocytes, and, in some embodiments, a portion of the population ofprogenitor cells present in skeletal muscle are capable of beingdifferentiated in vitro into genuine brown adipocytes. The presentdisclosure provides methods for expanding BAT progenitor cell culturesand methods for differentiating BAT progenitor cells into genuine BATcells, including methods for differentiating previously sorted cells inan adipogenic medium. In some embodiments, differentiation of sortedprogenitor cells into brown adipocytes can be performed using conditionsthat sustain white adipocyte differentiation or by use of agentsdetermined to promote differentiation of progenitors into brownadipocytes.

Some embodiments utilize the presence of UCP1, mitochondrialtranscription factor A (mtTFA), and/or PPARγ coactivator-1α (PGC-1α) aswell as one or more of the corresponding mRNAs, to identify BATprogenitor cells that have begun to at least partially differentiate.High metabolic rate or high levels of uncoupled respiration, glucoseutilization, fatty acid oxidation, or combinations of the foregoingcharacteristics with each other or other characteristics, can be used toidentify BAT progenitor cells that have begun to at least partiallydifferentiate. For purposes of this disclosure, BAT progenitor cellsthat have begun to at least partially differentiate into brownadipocytes are referred to as “differentiated brown adipocytes.”

As an example, cells determined to express the CD34 marker (i.e., CD34+cells) can be differentiated into brown adipocytes by culturing inDMEM-Ham's F-12 medium containing 0.86 μM insulin, 10 μg/ml transferrin,0.2 nM triiodothyronine, 1 μM rosiglitazone, 100 μM3-isobutyl-1-methylxanthine, 1 μM dexamethasone and 1%penicillin-streptomycin. Other agents may also be used to promotedifferentiation of progenitor cells into brown adipocytes. In someembodiments, agents identified according to the teachings of thisdisclosure are used to promote differentiation of progenitor cells intobrown adipocytes. In some embodiments, differentiated brown adipocytesexhibit high levels of UCP1 expression, high levels of uncoupledrespiration, and/or high metabolic rate.

The present disclosure provides methods for increasing UCP1 mRNAexpression in BAT progenitor cells, differentiated brown adipocytes, orboth. For example, agents such as cell-permeating cAMP derivatives andperoxisome-proliferator-activated receptor-γ (PPAR-γ) agonists can beused to increase UCP1 mRNA expression in BAT progenitor cells,differentiated brown adipocytes, or both. Enhanced UCP1 expression canbe determined by methods known in the art, including measurement of UCP1mRNA by quantitative RT-PCR. Exemplary primers for use in RT-PCRanalysis of UCP1 mRNA are provided as SEQ ID NOS: 1-4 and 11-12.

BAT progenitor cells exposed to adipogenic media can contain higherlevels of UCP1 mRNA than BAT progenitor cells that are not exposed toadipogenic media. Cyclophilin mRNA levels can serve as a normalizingvalue (reflecting the number of cells or the total amount of RNA) forevaluating the abundance of UCP1 mRNA in a cell. In some embodiments,UCP1 mRNA levels in BAT progenitor cells not exposed to adipogenic mediaare not detectable using RT-PCR while UCP1 mRNA levels in differentiatedbrown adipocytes is detectable and can be normalized to cyclophilin mRNAlevels. As a comparative measure of UCP1 expression, UCP1 mRNA levels indifferentiated brown adipocytes can be compared to UCP1 mRNA levels incultured mouse brown adipocytes. The present disclosure provides UCP1mRNA levels in differentiated brown adipocytes of about 25% of the UCP1mRNA levels in cultured mouse brown adipocytes, while in otherembodiments the UCP1 mRNA level is about 25±10% or from about 15% toabout 30% of the UCP1 mRNA levels in cultured mouse brown adipocytes.The present disclosure contemplates UCP1 mRNA levels in differentiatedbrown adipocytes in a range of from about 5% to about 100% of the UCP1mRNA levels in cultured mouse brown adipocytes. In some embodiments, theUCP1 mRNA levels can be in excess of 100% of the UCP1 mRNA levels incultured mouse brown adipocytes.

Differentiated brown adipocytes can contain significantly higher levelsof UCP1 mRNA than cells in same-species or same-individual adultskeletal muscle biopsies. In addition, the quantity of UCP1 protein in adifferentiated brown adipocyte can be approximately equal to thequantity of UCP1 protein in same-species or same-individual fetal BAT.The present disclosure contemplates UCP1 mRNA levels in humandifferentiated brown adipocytes being approximately equivalent to UCP1mRNA levels in human brown adipocytes in vivo. In some embodiments theUCP1 mRNA level in a human differentiated brown adipocyte can be in arange from about 1% to many times greater than UCP1 mRNA levels in humanbrown adipocytes in vivo.

The present disclosure provides methods for increasing UCP1 mRNA levelsin BAT progenitor cells, differentiated brown adipocytes, or both. Insome embodiments, the methods provide for selectively increasing UCP1mRNA levels in BAT progenitor cells, differentiated brown adipocytes, orboth. PPARγ agonists can stimulate UCP1 mRNA production in both skeletalmuscle and differentiated brown adipocytes. For example, in someembodiments, the PPARγ agonist rosiglitazone selectively stimulates UCP1mRNA production in skeletal muscle or in differentiated brownadipocytes. Cell-permeating cAMP derivatives can stimulate UCP1 mRNAproduction in both skeletal muscle and in differentiated brownadipocytes. For example, in some embodiments the cell-permeating cAMPderivative 8-bromo-cAMP selectively stimulates UCP1 mRNA production inskeletal muscle or in differentiated brown adipocytes while in someembodiments the cell-permeating cAMP derivative(4-chlorophenylthio)-cAMP selectively stimulates UCP1 mRNA production inskeletal muscle or in differentiated brown adipocytes.

Mitochondrial transcription factor A (“mtTFA”) andperoxisome-proliferator-activated receptor-γ coactivator-1α (“PGC-1α”)are involved in the control of mitochondriogenesis. Differentiated brownadipocytes can contain large amounts of mtTFA, PGC-1α, or both. Thepresent disclosure provides differentiated brown adipocytes havingsignificantly increased levels of mtTFA mRNA, PGC-1α mRNA, or both, ascompared to undifferentiated BAT progenitor cells. Mitochondrial markercytochrome oxidase IV (COX IV) is involved with the mitochrondrialrespiratory chain. The present disclosure provides differentiated brownadipocytes having significantly increased levels of COX IV mRNA ascompared to undifferentiated BAT progenitor cells.

Differentiated brown adipocytes according to some embodiments have highlevels of uncoupled respiration and/or high metabolic rate. Uncoupledrespiration can occur when protons leak across the inner mitochondrialmembrane rather than passing through the adenosine triphosphate synthase(“ATP Synthase”) enzyme to drive production of adenosine triphosphate(“ATP”). The energy released by the proton movement in theelectrochemical proton gradient across the membrane is dissipated asheat, rather than in the process of making ATP. Uncoupled respirationcan be measured as a function of the portion of cellular respiration(e.g., oxygen consumption) that occurs independently of ATP formation byATP Synthase. For example, oxygen consumption in the electron transportchain of oxidative phosphorylation in the presence of oligomycin, whichblocks the function of ATP Synthase, provides a measure of uncoupledrespiration.

The present disclosure provides differentiated brown adipocytes havingsignificantly increased levels of uncoupled respiration as compared toundifferentiated BAT progenitor cells. In some embodiments, the presentdisclosure provides differentiated brown adipocytes having levels ofuncoupled respiration of about 50% of total respiration. Someembodiments exhibit uncoupled respiration at levels in a range of fromabout 20% to about 50% of total respiration. Using the level ofuncoupled respiration in adult white adipocytes as a standard forcomparison, some embodiments exhibit uncoupled respiration in a range offrom about 1.5 to about 3.5 times greater than in adult whiteadipocytes. In some embodiments, the level of uncoupled respiration isabout 2.5 times greater than in adult white adipocytes. The presentdisclosure provides, among other things, differentiated brown adipocytesthat are equipped to metabolize glucose, oxidize fatty acids, anddissipate energy as heat via uncoupling of oxidative phosphorylation.

The present disclosure provides conditions and agents (e.g., compounds,proteins, biologicals, and the like) that promote the differentiation ofBAT progenitor cells to brown adipocytes, both in vitro and in vivo. Insome embodiments, the differentiation-promoting agent is: a PPARγactivator, modulator, or inhibitor (e.g., rosiglitazone), a PPARαactivator or modulator (e.g., GW9578), a PPARδ activator or modulator(e.g., GW501516 or GW0742), a dual PPARα and PPARδ activator ormodulator, a pan-PPAR (α, β, γ) activator or modulator (e.g., GW4148), aPDE4 inhibitor (e.g., rolipram or IBMX), a PDE7 inhibitor (e.g., BMS586353 or BRL 50481 or IBMX), a NRIP1 (RIP140) inhibitor, a PTENinhibitor (e.g., potassium bisperoxo (bipyridine) oxovanadate ordipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate), anα₁-adrenergic full or partial agonist (e.g., phenylephrine orcirazoline), an RxRα activator or modulator (e.g., LGD1069 (Targretin)or 9-cis retinoic acid), a PGC-1α activator, a PGC-1β inhibitor oractivator, adiponectin or an activator of adiponectin receptor AdipoR1and/or AdipoR2, an NOS inhibitor or activator (e.g.,2-Ethyl-2-thiopseudourea or NG-nitro-L-arginine methyl ester (L-NAME) oradenosine), a Rho kinase-ROCK inhibitor (e.g., fasudil), BDNF, amonoamine oxidase (MAO) A inhibitor and/or a MAO B inhibitor (e.g.,isocarboxazid, moclobemide, selegiline), an activator of SRC, aninhibitor of EGFR (e.g., erlotinib or ZD1839-gefinitib or Argosprotein), an inhibitor of FAAH (e.g., URB597), an inhibitor of MAPK 1(e.g., PD98059) or 2 (e.g., PD98059) or 4 or 5 or 7 or 8 (e.g.,PD98059), an inhibitor of CDK9 (e.g.,1,5,6,7-Tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-onehydrochloride), a TGR5 agonist (e.g., oleanolic acid), an AMPK activator(e.g., AICAR), BMP-7, an mTOR inhibitor (e.g., rapamycin), an adenylatecyclase activator (e.g., forskolin), or combinations of any of theforegoing.

In some embodiments, treatment of a subject, including a human subject,with rosiglitazone results in an increase in the production of UCP1 mRNAin the subject's skeletal muscle. Treatment of subjects withrosiglitazone can, in some embodiments, induce the appearance ordifferentiation of brown adipocytes in skeletal muscle, enhanceexpression of the UCP1 gene in existing brown adipocytes in skeletalmuscle, or both. For example, in some embodiments the appearance ordifferentiation of brown adipocytes in skeletal muscle can be induced ina subject suffering from a metabolic disease. The brown adipocytes canprovide a glucose sink with high mitochondrial and cellular respirationand fatty acid oxidation rates, dissipating energy as heat (uncoupledoxidative phosphorylation). The subject metabolic rate can be enhanced,and a decrease in body weight can be induced. Induction of theappearance or differentiation of brown adipocytes can also yieldimprovements in insulin sensitivity, blood glucose homeostasis andcardiovascular disease risk factors.

The present disclosure also provides assays that allow theidentification of agents (e.g., compounds, proteins, biologicals, andthe like) that promote the differentiation of BAT progenitor cells intobrown adipocytes and/or induce the expression of the UCP1 gene in vitro,in vivo, or both. Such agents can be identified by screening compounds,proteins, biologicals, and the like. For example, in some embodimentsisolated CD34+ cells can be used to screen agents for the ability toinduce expression of the UCP1 gene and/or differentiation of the CD34+cells into brown adipocytes. Agents identified in this manner can beused for a variety of research, diagnostic and therapeutic purposes,including, for example, treatment of metabolic diseases such as obesity,type 2 diabetes, insulin-resistance, dyslipidemia, and the like. In someembodiments, an agent identified by an assay according to the presentdisclosure is optimized for improvement of its physico-chemical and/orpharmacokinetics properties.

Expression of UCP1, mtTFA, PGC-1α, and/or COX IV in BAT progenitor cellsin vitro and in vivo can be enhanced according to methods provided inthe present disclosure. In some embodiments, exposure to adipogenicmedia can be used to stimulate increased expression of UCP1, mtTFA,PGC-1α, and/or COX IV in BAT progenitor cells. Agents such as a PPARγactivator, modulator or inhibitor (e.g., rosiglitazone), a PPARαactivator or modulator (e.g., GW9578), a PPARδ activator or modulator(e.g., GW501516 or GW0742), a dual PPARα and PPARδ activator ormodulator, a pan-PPAR (α, β, γ) activator or modulator (e.g., GW4148), aPDE4 inhibitor (e.g., rolipram or IBMX), a PDE7 inhibitor (e.g., BMS586353 or BRL 50481 or IBMX), a NRIP1 (RIP140) inhibitor, a PTENinhibitor (e.g., potassium bisperoxo (bipyridine) oxovanadate ordipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate), anα1-adrenergic full or partial agonist (e.g., phenylephrine orcirazoline), an RXRα activator or modulator (e.g., LGD1069 (Targretin)or 9-cis retinoic acid), a PGC-1α activator, a PGC-1β inhibitor oractivator, adiponectin or an activator of adiponectin receptor AdipoR1and/or AdipoR2, an NOS inhibitor or activator (e.g.,2-Ethyl-2-thiopseudourea or NG-nitro-L-arginine methyl ester (L-NAME) oradenosine), a Rho kinase-ROCK inhibitor (e.g., fasudil), BDNF, amonoamine oxidase (MAO) A inhibitor and/or a MAO B inhibitor (e.g.,isocarboxazid, moclobemide, selegiline), an activator of SRC, aninhibitor of EGFR (e.g., erlotinib or ZD1839-gefinitib or Argosprotein), an inhibitor of FAAH (e.g., URB597), an inhibitor of MAPK 1(e.g., PD98059), or 2 (e.g., PD98059) or 4 or 5 or 7 or 8 (e.g.,PD98059), an inhibitor of CDK9 (e.g.,1,5,6,7-Tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-onehydrochloride), a TGR5 agonist (e.g., oleanolic acid), an AMPK activator(e.g., AICAR), BMP-7, an mTOR inhibitor (e.g., rapamycin), an adenylatecyclase activator (e.g., forskolin) or combinations thereof can also beused to stimulate increased expression of UCP1, mtTFA, PGC-1α, and/orCOX IV in BAT progenitor cells.

EXAMPLES

Aspects of the present teachings may be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1 Sorting and Differentiation of Muscle Vascular Cells

In fetal skeletal muscle, CD34 and CD146 were found, byimmunohistochemistry, to be expressed at the surface of endothelialcells and pericytes, respectively, although CD34 was also expressed bycells scattered in the inter-myofibrillar space. FIG. 1(A) shows a smallvessel longitudinal section in which CD146+ pericytes (green) surroundCD34+ endothelial cells (red). A similar distribution of CD34+ andCD146+ cells was observed in adult skeletal muscle.

Vascular cells from seven independent fetal muscles (16-24 weeks ofgestation) were sorted using multi-color fluorescence-activated cellsorting (FACS). Hematopoietic (CD45+) cells were first gated out, aswere myogenic progenitors (CD56+). Then, endothelial cells(CD34+/CD146−) and pericytes (CD34−/CD146+) were sorted. TheCD34+/CD146−/CD45−/CD56− are designated thereafter as CD34+ cells andthe CD34−/CD146+/CD45−/CD56− as CD146+ cells. FIG. 1(B) showsCD34+/CD146− and CD34−/CD146+ cell purification. Dissociated cells werestained with PE-anti-CD34, FITC-anti-CD146, PE-Cy7-anti-CD56 andAPC-Cy7-anti-CD45 antibodies and run on a FACS Aria cell sorter.Following exclusion of CD45+ and CD56+ cells (left panels), cells insidethe CD34+ or CD146+ gates were isolated. The CD34+ cells amounted to8±1% of the starting fetal muscle cell population.

FIG. 1(C) shows RT-PCR analysis on CD34+/CD146−/CD45−CD56− (CD34),CD34−/CD146+/CD45−/CD56− (CD146) and total non-sorted cells. Actin mRNAwas measured as a control. The CD34+ cells were shown not to becontaminated by detectable CD45+ hematopoietic or CD56+ myogenic cells.

Sorted cells were grown 4-6 days in EGM2 medium and 8-12 days in theadipogenic medium described under Materials and Methods. Theseconditions sustain white adipocyte differentiation in WAT primarycultures. FIG. 2 shows CD34+ (FIG. 2(A)) and CD146+ (FIG. 2(B), FIG.2(C)) cells in primary cultures (PC) and CD34+ (FIG. 2(D)) cellsexpanded in culture up to passage 3 (P3). Virtually all sorted fetalmuscle CD34+ cells differentiated into adipocyte-like multilocular cells(FIG. 2(A), 2(D)). It is noteworthy that in cell culture, themultilocular structure is shared by white and brown adipocytes. Incontrast, fetal muscle CD146+ cells grew very slowly under theconditions described above. They did not reach cell confluence anddisplayed a pericyte-like appearance characterized by a large size,spread out shape and irregular borders (FIGS. 2(B) and (C)). Occasionalmultilocular cells could be detected (FIG. 2(C)) The morphology of CD34+cells expanded in culture for up to 3 passages (4 weeks) under theconditions described above was similar to that observed in primaryculture, although the size of mature adipocytes was smaller (FIG. 2(D)).

Example 2 UCP1 Expression in Cultivated CD34+ Cells

The remarkable adipocyte-like differentiation of fetal muscle CD34+cells was an incentive for further characterization. Strikingly,quantitative RT-PCR revealed a high level of UCP1 mRNA in these cells.FIG. 2(E) shows quantitative RT-PCR determination of UCP1 (emptycolumns) and leptin (gray columns) mRNA expression in CD34+ cells inprimary culture (PC) or expanded up to passage 3 (P3). The mean UCP1mRNA level normalized to cyclophilin A was 1797±510 arbitrary units(i.e., ±s.e.m. of arbitrary values normalized using the correspondingcyclophilin A values; n=4-7), corresponding to a cycle threshold (Ct) of22 for 25 ng of cDNA in the assay.

For comparison, the mean UCP1 mRNA level normalized to cyclophilin A inmouse brown adipocytes differentiated in culture was 7715±2649 (n=10)arbitrary units. Therefore, the level of UCP1 mRNA in human CD34+ cellsamounted to almost one fourth of that in mouse brown adipocytes inculture. Human fetus BAT was not be used as a positive control forquantitative RT-PCR analysis because the risk of RNA degradation washigh due to the time elapsed after the termination of the pregnancy. Theamplicon was cloned and sequenced and found to be 100% identical tohuman UCP1. In fetal muscle CD34+ cells expanded up to passage 3 a highUCP1 mRNA expression, amounting to 43% of that detected in primarycultured cells, was still observed. UCP1 mRNA expression was notdetected in non-differentiated fetal muscle CD34+ cells or in CD146+cells in primary culture. The level of leptin mRNA was 9.9±5.5 and 71±52arbitrary units in primary cultured and expanded cells, respectively(FIG. 2E).

Example 3 Additional Phenotyping of the CD34+ Cells

To better characterize the gene expression pattern of the fetal muscleCD34+ cells expanded in culture a gene chip analysis was performed. Thelevels of expression of several representative gene mRNAs withsignificant Detection P-Values (p<0.01) are shown in Table 1 andcompared with those in human muscle biopsies. The following proteinmRNAs were chosen: UCP1 as a reference gene, mitochondrial transcriptionfactor A (mtTFA) and peroxisome-proliferator-activated receptor (PPARγ)and PPARγ coactivator-1α (PGC-1α), which are involved in the control ofthermogenesis and mitochondriogenesis, enzymes of the mitochondrialrespiratory chain succinate dehydrogenase (SDH) and cytochrome oxidaseIV (COX IV), enzymes of the fatty acid degradation pathway, carnitinepalmitoyltransferase 1B (CPT1B), acyl-CoenzymeA dehydrogenases longchain (ACAD) and C-4 to C-12 straight chain (ACADM), and the skeletalmuscle markers myogenin, myogenic factor 5 (Myf5) and myogenicdifferentiation1 (MyoD1). Cidea, which is highly expressed in BAT andmay act as a suppressor of UCP1 activity [16], was chosen as a BATmarker. The Genbank accession numbers of these genes are shown in thesupplemental data.

TABLE 1 Accession Human muscle mRNA No. CD34+ cells biopsies UCP1NM_021833 94 n.s. mTFA NM_003201.1 413 205 PPARγ NM_138712.2 3326 84PGC-1α NM_013261.2 137 619 COX IV NM_001861.2 13′082 13′407 SDHNM_003000.1 2390 5187 CPT1B NM_004377.2 99 639 ACAD NM_032169.3 1032 141ACADM NM_000016.2 599 1640 Myogenin NM_002479.3 n.s. 267 Myf5 NM_05593n.s. 21 MyoD1 NM_002478 n.s. 12 Cidea NM_198289.1 337 n.s.

The data in Table 1 are expressed as the average Illumina signal. TheDetection P-Values are <0.01. The following abbreviations are used:n.s., not significant; mtTFA, mitochondrial transcription factor A;PPARα, peroxisome-proliferator-activated receptor-γ; PGC-1α, PPARγcoactivator-1α; COX IV, cytochrome oxidase IV; SDH, succinatedehydrogenase; CPT1B, carnitine palmitoyltransferase 1B; ACAD,acyl-CoenzymeA dehydrogenases long chain; ACADM, C-4 to C-12 straightchain; Myf5, myogenic factor 5; MyoD1, myogenic differentiation 1.

UCP1 was significantly expressed in fetal muscle-expanded CD34+ cellsbut not in adult muscle biopsies (for which p=0.12). The levels of mRNAexpression of the selected genes in expanded CD34+ cells from fetalmuscle were comparable with those of the adult muscle biopsies with theexceptions of PGC-1α and CPT1B mRNAs (which were about 5-fold lessexpressed in the cells) and of the PPARγ and ACAD mRNAs (which were 40-and 7-fold less expressed, respectively in the muscle biopsies). Themuscle markers myogenin, Myf5 and MyoD1 mRNA were significantlyexpressed in the muscle but not in the cells whereas the BAT markerCidea mRNA was expressed in the cells but not in the muscle. Noβ₃-adrenoceptor mRNA could be detected in the gene chip analysis. It isnoteworthy, however, that β₃-adrenoceptor mRNA was detected byquantitative RT-PCR (arbitrary value 0.084±0.044 with cyclophilin A as areference; n=4) in fetal muscle CD34+ cells in primary culture.Measurements of mtTFA, PGC1-α and COX IV were also performed byquantitative RT-PCR to confirm the gene chip data with a differenttechnique. The results were confirmatory, showing that fetal muscleCD34+ cells in primary culture express high levels of mtTFA, PGC1-α andCOX IV mRNA [amounting to 306±117, 385±294, and 23,400±10,300 arbitraryunits (n=3-4), respectively], using cyclophilin A as a reference.

The UCP1 protein, as assessed by Western blotting with an anti-mouseantibody cross-reacting with human UCP1 (80% identity), was as abundantin primary cultured fetal muscle CD34+ cells as in fetal BAT. FIG. 2(F)shows representative Western blot analysis of UCP1 and glyceraldehydephosphate dehydrogenase (GAPDH) proteins in tissue or whole cellextracts. Interscapular BAT of a 19-week fetus (Lane 1), CD34+ cells inprimary culture (Lane 2), and skeletal muscle of an adult human (Lane 3)are shown. 25 μg of protein was loaded into each lane.

Example 4 Uncoupling of Oxidative Phosphorylation

To get insight into the possible function of UCP1 in muscle-derivedcells, mitochondrial respiration of isolated cultured human fetal muscleCD34+ cells and human adult white adipocytes was compared. Basalrespiration was defined as the antimycin A-sensitive oxygen consumption.Uncoupled respiration (proton leak) was defined as the percentage ofbasal respiration insensitive to the ATP synthase blocker oligomycin.

FIG. 3(A) shows uncoupling of mitochondrial respiration in isolatedfetal muscle CD34+ cells and in human adult white adipocytes grown inprimary culture and freshly trypsinized. The results are means±s.e.m;*p<0.05, n=3. The ratios of uncoupled to total respiration were 47±12%and 19±2% in human fetal muscle CD34+ cells and adult white adipocytes,respectively.

Example 5 Modulation of UCP1 Expression in Cultured CD34+ Cells

UCP1 mRNA expression in fetal muscle CD34+ cells could be modulated bydrug treatment. Cell-permeating cAMP derivatives strongly stimulated (7to 8-fold) UCP1 mRNA expression in both primary cultured and expandedcells. The effects of cAMP derivatives, 8-bromo-cAMP, 0.25 mM or(4-chlorophenylthio)-cAMP, 0.25 mM (cAMP) on UCP1 mRNA expression inCD34+ cells in primary culture (PC) or expanded up to passage 3 (P3) areshown in FIG. 3(B). All the cells were grown for 4-6 days in EGM2 mediumand then placed for 8-12 days in the adipogenic medium described underMaterials and Methods. The results are means±s.e.m. of arbitrary valuesnormalized using the corresponding cyclophilin A values. They areexpressed in % of their respective untreated (control) values consideredas 100% (*p<0.05, n=3-6).

Rosiglitazone, a PPARγ agonist, had no effect in primary culture cellsbut strongly stimulated (8-fold) UCP1 mRNA expression in expanded cells.The effects of rosiglitazone (Rosi) 1 μM on UCP1 mRNA expression inCD34+ cell PC or P3 are shown in FIG. 3(C). The results are expressed asin FIG. 3(B) (**p<0.01, n=4-7).

Example 6 Muscle Specificity and Persistence Throughout Life of HumanBrown Adipocyte Progenitors

The derivation of UCP1-expressing cells from human fetal muscle raisedthe question of the restriction of brown adipocyte progenitors to thistissue and to the fetal stage. To address this issue, CD34+ cellspurified by FACS from human fetal pancreas, lung and liver were culturedunder the same adipogenic conditions as fetal muscle CD34+ cells. Thesorted cells grew slowly and only a small proportion of them becamemultilocular. UCP1 mRNA was not expressed in pancreas or lung cells;however, a minor expression was measured in liver cells, which amountedto 2% of that detected in fetal muscle CD34+ cells (not shown).

CD34+ cells sorted from 4 adult (50-78 years) human skeletal musclesamples, grown in primary culture (PC) under adipogenic conditions, alsodifferentiated into multilocular cells. These cells were interspersedwith other types of cells, some of them containing small lipid droplets(FIG. 4(A)). The level of UCP1 mRNA (370±132 arbitrary units) was 21% ofthat detected in primary cultured fetal muscle CD34+ cells. In contrast,leptin expression (75±69 arbitrary units) was 7.6-fold higher than infetal cells. FIG. 4(B) shows quantitative RT-PCR determination of UCP1(empty column) and leptin (gray column) mRNA expression. All the cellswere grown for 4-6 days in EGM2 medium and then placed for 8-12 days inthe adipogenic medium described under Materials and Methods. The resultsare the mean±s.e.m. of arbitrary values normalized to the correspondingcyclophilin A values (n=4-5). CD34+ cells sorted from 4 adult (45-55years) human WAT samples were also grown in primary culture (PC) underadipogenic conditions. They became partially multilocular (FIG. 4(C)),but did not express UCP1 mRNA.

Example 7 Detection of UCP1 mRNA Expression in Human Muscle and Effectof Rosiglitazone In Vivo

Brown adipocyte progenitors of adult human skeletal muscle candifferentiate in vivo and give rise to UCP1 expressing cells. Thepresence of UCP1 mRNA in the adult human skeletal muscle was trackedusing a high sensitivity RT-PCR technique and, in fact, low levels ofUCP1 mRNA were detected in the rectus abdominus muscle of 10 leansubjects (UCP1/cyclophilinA ratio: 24±9). The PCR-amplified fragment wassequenced and found to be 100% identical to human UCP1. The UCP1 mRNAlevel in adult human muscle was 75-fold lower than that in fetal muscleCD34+ cells in culture.

Since the PPARγ agonist rosiglitazone was a strong inducer of UCP1 mRNAexpression in muscle CD34+ cells in culture, the effect of this compoundin vivo in humans was investigated. Vastus lateralis muscle biopsiesfrom 7 obese patients with type 2 diabetes mellitus treated for themanagement of their metabolic syndrome with rosiglitazone were used. Thebiopsies were obtained before and after 8 weeks of treatment withrosiglitazone (2×4 mg per day). The treatment with rosiglitazoneresulted in a significant improvement of the patients' insulinresistance and diabetes. In that study rosiglitazone, concomitantly withthe improvement in insulin sensitivity, increased the level ofexpression of UCP1 in muscle by about 1.6-fold. FIG. 5 shows thequantitative RT-PCR determination of UCP1 mRNA expression. The resultsare the means±s.e.m. of arbitrary values normalized using thecorresponding cyclophilin A values (n=7, *p<0.05 vs. control). Since theRT-PCR conditions used were different, the arbitrary values of thisfigure do not provide a direct comparison to those of FIGS. 2-4.

In FIG. 5, showing UCP1 mRNA levels in skeletal muscle biopsies from apatient group (n=7), “control” corresponds to levels before treatment,and “Rosi” corresponds to levels after treatment (8 weeks) withrosiglitazone. Being a longitudinal study the effect of rosiglitazone onUCP1 levels in each individual (comparison of individual values for the“control”-before and “Rosi”-after conditions) were determined. Startingwith 25 ng cDNA (produced by reverse transcription of RNA) the thresholdof detection (Ct) during real-time PCR was about 22 for UCP1 and about18 for cyclophilin. The effect of rosiglitazone (UCP1 level at end oftreatment vs. before treatment) were as follows:

Patient 1: 50% increase (to 150%, control=437, Rosi=652 arbitrary units)

Patient 2: no change (to 100%, control=444, Rosi=453 arbitrary units)

Patient 3: 80% increase (to 180%, control=378, Rosi=677 arbitrary units)

Patient 4: 180% increase (to 280%, control=260, Rosi=730 arbitraryunits)

Patient 5: 8% increase (to 108%, control=553, Rosi=600 arbitrary units)

Patient 6: 310% increase (to 410%, control=135, Rosi=556 arbitraryunits)

Patient 7: 10% increase (to 110%, control=128, Rosi=142 arbitrary units)

Strong effects of rosiglitazone, varying between 1.5- and 4.1-fold, wereobserved in 4 out 7 patients. This result suggests that rosiglitazoneinduced the appearance of brown adipocytes and/or enhanced theexpression of the UCP1 gene in existing brown adipocytes in the skeletalmuscle of the patients. This effect of the PPARγ agonist may play a keyrole in the therapeutic effect of this agent as an insulin-sensitizer.

Example 8 Screening of Potential Modulators of the Human UCP1Promoter/Enhancer Region

The identified and isolated CD34+ cells can be used as a tool toidentify agents (compounds, proteins, biologicals, and the like) thatinduce the differentiation of these cells into brown adipocytes ormodulate the expression of UCP1.

For this purpose a large region (6 kb) of DNA upstream (in 5′) of thetranscription start site of the human UCP1 gene (containing thepromoter/enhancer region) has been cloned into a reporter/MAR GFP (GreenFluorescent Protein) or luciferase. This construct has been used totransfect CD34+ cells, and the cells grown in multiwell plates andscreened for agents that increase the fluorescence (GFP) or luminescence(luciferase) of the cells, reflecting induction of gene expression (andthus increased UCP1 expression). This allows the identification ofagents that can enhance the differentiation of CD34+ cells into brownadipocytes and/or the expression of UCP1 by enhancing the transcriptionof the UCP1 gene and/or by enhancing the translation of the UCP1transcript, and/or by stabilizing the UCP1 transcript or protein.

For example, a PPARγ modulator or activator like rosiglitazone can beused to promote the differentiation of CD34+ progenitor cells into brownadipocytes (FIGS. 3(C) and 5). Another example is the use of cAMPderivatives like, 8-bromo-cAMP and/or (4-chlorophenylthio)-cAMP (FIG.3(B)) or protein kinase A (PKA) activators or phosphodiesteraseinhibitors. Another example is the use of triiodothyronine (T3), otherthyroid hormones, agonists or modulators of the thyroid hormonereceptors TRα and/or TRβ. Another example is to use β-adrenergicagonists like isoproterenol (pan-agonist) or specific β₁-, β₂-,β₃-agonists or modulators. Another is the use of modulators of thecandidate receptors revealed by gene chip studies or of target genes inthe signaling pathway downstream these receptors.

Example 9 Gene Chip Studies

Gene chip studies were performed to identify molecular pathways thatplay a role in the differentiation of CD34+ progenitor cells into brownadipocytes and/or the induction of the expression of UCP1. CD34+ cellswere isolated from human skeletal muscle biopsies, and were used in twostudies: (1) cAMP study: CD34+ cells were differentiated as described inMaterials (Control) plus addition of vehicle (Control 1 sample) or cAMP(cAMP sample); and (2) Rosiglitazone study: CD34+ cells weredifferentiated as described in Materials except that rosiglitazone wasomitted from the adipogenic medium (Control 2 sample). Rosiglitazone wasadded only to the second sample (Rosiglitazone sample) in this study.

We have found that these compounds promote the differentiation of CD34+cells into brown adipocytes and the expression of UCP1 (see FIGS. 3(B),(C)).

Total RNA was purified from these cells, and transcriptional profileswere assessed with Illumina Human WG-6 BeadChip (Expression Analysis,Inc., Durham, N.C.). Results were analyzed with Ingenuity PathwayAnalysis 7.0 (trial version). These results were used to determine whatmolecular pathways are involved in the differentiation of CD34+ cellsinto brown adipocytes, and, more importantly, what molecular targets canbe used for the development of agents that promote the appearance ofbrown adipocytes and the expression of UCP1.

This work showed that the following actions/agents should promote brownadipocyte development: a PPARγ activator, modulator or inhibitor (e.g.,rosiglitazone), a PPARα activator or modulator (e.g., GW9578), a PPARδactivator or modulator (e.g., GW501516 or GW0742), a dual PPARα andPPARδ activator or modulator, a pan-PPAR (α, β, γ) activator ormodulator (e.g., GW4148), a PDE4 inhibitor (e.g., rolipram or IBMX), aPDE7 inhibitor (e.g., BMS 586353 or BRL 50481 or IBMX), a NRIP1 (RIP140)inhibitor, a PTEN inhibitor (e.g., potassium bisperoxo (bipyridine)oxovanadate or dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl)oxovanadate), an α1-adrenergic full or partial agonist (e.g.,phenylephrine or cirazoline), an RXRα activator or modulator (e.g.,LGD1069 (Targretin) or 9-cis retinoic acid), a PGC-1α activator, aPGC-1β inhibitor or activator, adiponectin or an activator ofadiponectin receptor AdipoR1 and/or AdipoR2, an NOS inhibitor oractivator (e.g., 2-Ethyl-2-thiopseudourea or NG-nitro-L-arginine methylester (L-NAME) or adenosine), a Rho kinase-ROCK inhibitor (e.g.,fasudil), BDNF, a monoamine oxidase (MAO) A inhibitor and/or a MAO Binhibitor (e.g., isocarboxazid, moclobemide, selegiline), an activatorof SRC, an inhibitor of EGFR (e.g., erlotinib or ZD1839-gefinitib orArgos protein), an inhibitor of FAAH (e.g., URB597), an inhibitor ofMAPK 1 (e.g., PD98059), or 2 (e.g., PD98059) or 4 or 5 or 7 or 8 (e.g.,PD98059), an inhibitor of CDK9 (e.g.,1,5,6,7-Tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-onehydrochloride), a TGR5 agonist (e.g., oleanolic acid), an AMPK activator(e.g., AICAR), BMP-7, an mTOR inhibitor (e.g., rapamycin), and adenylatecyclase activator (e.g., forskolin), or combinations of any of theforegoing.

Materials & Methods

All organic and inorganic chemicals of analytical or molecular biologygrade were purchased from Sigma Chemical Co. (St Louis, Mich.) and GibcoBRL (New York, N.Y.).

Human Tissues

Human fetal tissues were obtained anonymously, following spontaneous,voluntary or therapeutic terminations of pregnancy, from Magee WomenHospital, University of Pittsburgh, in compliance with the InstitutionalReview Board protocol. Developmental age (16 to 24 weeks of gestation)was estimated by measuring foot length. Informed consent to the use offetal tissues was obtained from the patients in all instances. Adulthuman discarded abdominal subcutaneous WAT, originating from 45-55 yearold patients undergoing plastic surgery performed one year after gastricbypass, was kindly provided by Dr Peter Rubin (Division of PlasticSurgery, University of Pittsburgh). The adult skeletal muscle tissueused for cell sorting was obtained post mortem from 50-78 year-olddonors. The adult skeletal muscle used for the first group of RT-PCRstudies was obtained from the rectus abdominus during surgery for eitherlap banding, inguinal hernia or hysterectomy of 10 lean male and femalesubjects. All subjects agreed to donate muscle samples during theiroperations and the protocol was approved by the Medical Ethical ReviewCommittee of Deakin University. The average ages were 45±3 years and theaverage body mass index was 22.2±0.8. The adult skeletal muscle used forthe second group of RT-PCR studies was obtained from the vastuslateralis of 7 obese type 2 diabetic male and female patients before andafter 8 weeks of treatment with rosiglitazone (2×4 mg per day). Theaverage age was 63±4 years and the average body mass index was 29.9±3.8.The complete clinical profile of the patients has been described in aprevious publication [18]. All subjects agreed to donate muscle samples,and the protocol was approved by the Medical Ethical Review Committee ofMaastricht University.

Mice

Animals were treated in accordance with the Centre Médical Universitaire(Genéve) institutional guidelines. They were housed individually andkept on a 12 h light-dark cycle in a temperature-controlled room at 24°C. They were allowed ad libitum access to water and a standardlaboratory chow. The interscapular BAT of 4- to 6-week-old male 129Sv/ev mice were excised and their precursor cells isolated and culturedas previously described [19].

Immunohistochemistry

Fresh fetal and adult tissues were gradually frozen by immersion inisopentane cooled in liquid nitrogen. Five- to 7-μm sections were cut ona cryostat (Microm HM 505 E), fixed with 50% acetone and 50% methanol,dried for 5 min at room temperature (RT), and then washed 3 times for 5min in phosphate-buffered saline. Non-specific binding sites wereblocked with 5% goat serum for 1 hour at RT. Sections were incubatedovernight at 4° C. with a CD34 mouse anti-human antibody (Serotech,1:50), then, after rinsing, for 1 hour at RT with a secondary goatanti-mouse biotinylated antibody (DAKO, 1:1000) and for 30 min at RTwith streptavidin-Cy3 (Sigma, 1:1000) or for 2 hours at RT with aconjugated CD146-Alexa 488 mouse anti-human antibody (Chemicon, 1:200).Nuclei were stained with 4′,6-diamino-2-phenylindole dihydrochloride(Molecular Probes, 1:2000) for 5 min at RT. An isotype-matched negativecontrol was performed with each immunostaining.

Flow Cytometry

The vascular cells of fetal skeletal muscle, pancreas, lung and liver aswell as of adult muscle and WAT were analysed by flow cytometry. Freshfetal or adult muscle as well as fetal pancreas, lung and liver tissueswere cut into small pieces with a scalpel in Dulbecco's Modified EagleMedium high glucose (DMEM) containing 20% fetal bovine serum (FBS), 1%penicillin-streptomycin (PS) and collagenases IA-S, II-S and IV-S (1mg/mL), then incubated at 37° C. for 75 min (fetal tissues) or 90 min(adult tissues) with constant stirring. Final cell dissociation wasachieved between ground glass slides. Cells were washed withphosphate-buffered saline and centrifuged for 5 min at 350 g. They wereresuspended in DMEM, 20% FBS, filtered at 100 μm, stained with Trypanblue and counted after dead cell exclusion. The WAT stroma vascularfraction was prepared by collagenase digestion according to Champigny etal. [20]. Cells (10⁵ for analysis and around 30·10⁶ for sorting) wereincubated with one of the following directly coupled mouse anti-humanantibodies: CD45-APC Cy7 (Santa Cruz Biotechnologies, 1:200), CD56-PECy7 (BD Pharmigen 1:100), CD34-PE (DAKO, 1:100) and CD146-FITC (Serotec,1:100) in 1 ml DMEM, 20% FBS, 1% penicillin-streptomycin, at 4° C. for15 min. After washing and centrifugation cells were incubated 30 minwith 7-amino-actinomycin D (7-AAD, BD Pharmigen, 1:100) for dead cellexclusion, filtered at 70 □m and run on a FACS Aria flow cytometer(Becton Dickinson). As negative controls, cell aliquots were incubatedwith isotype-matched mouse IgGs conjugated to APC Cy7 (BD Pharmigen,1:100), PE Cy7 (BD Pharmigen, 1:100), PE (Chemicon, 1:100) and FITC (USBiological, 1:100) under the same conditions.

Cell Culture

Cells were seeded at 2·10⁴ per cm² in 0.2% gelatin coated plates,cultured until confluency (4-6 days) at 37° C. in EGM2 medium (CambrexBio Science, Walkersville, Md.) and until differentiation (8-12 moredays) in a modification of the adipogenic medium described by Rodriguezet al. [21] consisting in DMEM-Ham's F-12 medium containing 0.86 μMinsulin, 10 μg/ml transferrin, 0.2 nM triiodothyronine, 1 μMrosiglitazone (GlaxoSmithKline, Research Triangle Park, N.C.), 100 μM3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone and 1%penicillin-streptomycin. For cell expansion studies, confluent cellsgrown in EGM2 medium only were detached by treatment with trypsin-EDTAfor 3-5 min at 37° C., and then split 1:3 and cultured as describedabove. Human white adipocytes in primary culture used in the oxymetrystudies were obtained as previously described [22].

RT-PCR

Total cell RNA was prepared using the kit NucleoSpin® RNAII (Clontech,Palo Alto, Calif.) or Extract-all solution (Eurobio, Courtaboeuf,France) and quantified by Biophotometry (Biophotometer, Eppendorf).Oligo-dT primed First strand cDNA were synthesized using theSuperscript™ II RNase H Reverse Transcription kit (Invitrogen, Carlsbad,Calif.) and oligo-dT primers or the High Capacity cDNA ReverseTranscription kit (Applied Biosystems, Foster City, Calif.) and randomprimers. Quantitative real-time PCR was performed using ABI rapidthermal cycler system, and a SYBR Green PCR master mix (AppliedBiosystems, Foster City, Calif.). Cyclophilin A was used as a control toaccount for any variations due to the efficiency of the reversetranscription. The upstream and downstream oligonucleotide primers werechosen on both sides of an intron to prevent amplification ofcontaminating genomic DNA.

The primers used for real time quantitative PCR in human cells and inmouse brown adipocytes are as follows:

hUCP1

Sense primer: 5′-CCTCACCGCAGGGAAAGAA-3′ (SEQ ID NO: 1)

Antisense primer: 5′-CTAACGACTGGAGGAGTGGCA-3′ (SEQ ID NO:2)

Amplicon position: 429-504.

Accession No.: NM_(—)021833.

mUCP1

Sense primer: 5′-CGATGTCCATGTACACCAAGGA-3′ (SEQ ID NO:3)

Antisense primer: 5′-TTGTGGCTTCTITTCTGCGA-3′ (SEQ ID NO:4)

Amplicon position: 996-1063.

Accession No.: NM_(—)009463.2.

hleptin

Sense primer: 5′-CCAAAACCCTCATCAAGACAATT-3′ (SEQ ID NO:5)

Antisense primer: 5′-AAGTCACCGGTTTGGACTTCA-3 (SEQ ID NO:6)

Amplicon position: 143-238.

Accession No.: BC069323.

hcyclophilin A

Sense primer: 5′-CATCTGCACTGCCAAGACTGA-3′ (SEQ ID NO:7)

Antisense primer: 5′-GCAAAGTGAAAGAAGGCATGAA-3′ (SEQ ID NO:8)

Amplicon position: 466-537.

Accession No.: NM_(—)203431.

mcyclophilin A

Sense primer: 5′-CAAATGCTGGACCAAACACAA-3′ (SEQ ID NO:9)

Antisense primer: 5′-CCATCCAGCCATTCAGTCTTG-3′ (SEQ ID NO:10)

Amplicon position: 343-412.

Accession No.: NM_(—)008907.

Primer used for real time quantitative PCR in human skeletal muscle areas follows:

hUCP1

Sense primer: 5′-TCCGGCTCCAGGTCCAA-3′ (SEQ ID NO:11)

Antisense primer: 5′-TGATTGTTCCCAGGACACCTTT-3′ (SEQ ID NO:12)

Amplicon position: 240-311.

Accession No.: NM_(—)021833.

hcyclophilin A

Sense primer: 5′-CATCTGCACTGCCAAGACTGA-3′ (SEQ ID NO:7)

Antisense primer: 5′-GCAAAGTGAAAGAAGGCATGAA-3′ (SEQ ID NO:8)

Amplicon position: 466-537.

Accession No.: NM_(—)203431.

Primers used for analytical PCR are as follows:

CD34

Sense primer: 5′-CATCACTGGCTATTTCCTGATG-3′ (SEQ ID NO:13)

Antisense primer: 5′-AGCCGAATGTGTAAAGGACAG-3′ (SEQ ID NO:14)

Amplicon position: 1172-1591.

Accession No.: M81104.

CD56

Sense primer: 5′-GTATTTGCCTATCCCAGTGCC-3′ (SEQ ID NO:15)

Antisense primer: 5′-CATACTTCTTCACCCACTGCTC-3′ (SEQ ID NO:16)

Amplicon position: 542-873.

Accession No.: BC014205.

CD45

Sense primer: 5′-CATGTACTGCTCCTGATAAGAC-3′ (SEQ ID NO:17)

Antisense primer: 5′-GCCTACACTTGACATGCATAC-3′ (SEQ ID NO:18)

Amplicon position: 940-1579.

Accession No.: Y00638.

CD146

Sense primer: 5′-AAGGCAACCTCAGCCATGTCG-3′ (SEQ ID NO:19)

Antisense primer: 5′-CTCGACTCCACAGTCTGGGAC-3′ (SEQ ID NO:20)

Amplicon position: 168-603.

Accession No.: M28882.

β-actin

Sense primer: 5-CCTCGCCTTTGCCGATCC-3′ (SEQ ID NO:21)

Antisense primer: 5′-GGAATCCTTCTGACCCATGC-3′ (SEQ ID NO:22)

Amplicon position: 25-229.

Accession No.: NM_(—)001101.

Arbitrary units were determined by normalizing target mRNA levels tocyclophilin mRNA levels (based on Cts), wherein the cyclophilin levelswere first divided by 100,000 for ease of reference. For example, aratio of target mRNA to cyclophilin mRNA of 0.01797 is expressed as1797.

Validation of the Human UCP1 Amplicon.

The PCR-amplified fragment was cloned into the pCR2.1-TOPO vectorthrough the TOPO-TA cloning system (Invitrogen, Carlsbad, Calif.) andpurification of color-selected colonies was performed using the QiaprepSpin Miniprep (Qiagen, Hilden, Germany). Sequences were determined witholigonucleotide M13 Reverse on the pCR2.1-TOPO vector using the AppliedBiosystem Big Dye sequencing kit on an ABI 3700 automated sequencer(Applied Biosystems, Foster City, Calif.).

Western Blots

Cultured cells were collected with a rubber policeman in 200 μl of RIPAbuffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% SDS,1:200 protease inhibitor cocktail (Sigma Chemical Co, St Louis, Mich.)and 50 mM Tris/HCl pH 8.0). Human BAT and skeletal muscle werehomogeneized in the above RIPA buffer. The protein content wasdetermined according to the technique of Lowry [23]. Western blots wereperformed as previously described [24]. The UCP1 protein was detectedusing a 1/500 diluted rabbit anti-mouse UCP1 polyclonal primary antibodygenerously provided by Dr B. Cannon (Stockholm, Sweden). This antibodyhad been raised against the C-terminal decapeptide of mouse UCP1, thatshares 80% identity with human UCP1 and 0 and 10% identities with humanUCP2 and UCP3, respectively. Glyceraldehyde phosphate dehydrogenase(GAPDH) protein was detected using a 1/5000 diluted mouse anti-mouseGAPDH monoclonal primary antibody (Chemicon International, Inc,Temecula, Calif.). 1/5000 diluted goat anti-rabbit or anti-mouseperoxidase-labelled secondary antibodies (Sigma-Aldrich, St. Louis, Mo.or Bio-Rad, Hercules, Calif.) were used. A SeeBlue® Plus 2 Pre-stainedStandard Ladder (Invitrogen, Carlsbad, Calif.) was used. Protein signalswere detected by chemiluminescence using a standard ECL kit anddeveloped on a Hyperfilm ECL film.

High-Resolution O₂ Consumption Measurement

Oxygen consumption was measured using a 2-injection chambersrespirometer equipped with a Peltier thermostat, Clark-type electrodes,and integrated electromagnetic stirrers (Oroboros® Oxygraph, Oroboros,Innsbruck, Austria). Measurements were performed at 37° C. withcontinuous stirring in 2 ml of DMEM F12, 10% new born calf serum. Underthese conditions, the serum provided the fatty acids necessary tosustain UCP1 uncoupling activity. Before each O₂ consumptionmeasurement, the medium in the chambers was equilibrated with air for 30min, and freshly trypsinized cells were transferred into therespirometer glass chambers. After observing steady-state respiratoryflux, ATP synthase was inhibited with oligomycin (0.25-0.5 mg/l) andcells were titrated with the uncoupler carbonyl cyanide3-chloro-phenylhydrazone up to optimum concentrations in the range of1-2 μM. The respiratory chain was inhibited by antimycin A (1 μg/ml).Oxygen consumption was calculated using DataGraph software (Oroborossoftware).

Gene Chip Analysis

The total RNA of fetal muscle CD34+ cells expanded in culture for up to3 passages (4 weeks) and of human muscle biopsies were prepared asdescribed above. The quality assurance measurements, the preparation ofthe cRNA targets and the gene chip analyses using Illumina Human WG-6BeadChip were performed by Expression Analysis, Inc. (Durham, N.C.).BeadStudio nonparametric methods were used for the computation ofDetection P-Values.

Statistical Analysis

Data are expressed as means±s.e.m. Significances were evaluated usingthe unpaired Student's t-test. A paired Student's t-test was used todetermine the effects of rosiglitazone on human skeletal muscle UCP1mRNA levels. Significances were set at p<0.05.

Cloning of the Human UCP1 Promoter/Enhancer Region:

To develop our screening strategy, the human UCP1 promoter/enhancer wassubcloned as follow:

A human BAC (bacterial artificial chromosome) clone #RP11-5K16, (AC108019) containing the human UCP1 (uncoupling protein-1)promoter/enhancer region, was obtained from the CHORI (Children'sHospital Oakland Research Institute) BAC-PAC resources service. Theselected promoter/enhancer region starts at position −25 upstream of the5′UTR (UnTranslated Region) of the human UCP1 gene (accession number:NM_(—)021833). Based on the human UCP1 gene initiation codon, the fullcloned promoter/enhancer sequence locates between position −149 and−6269.

Primer sets were designed to amplify either:

i) the full targeted promoter/enhancer region (6120 bp starting atposition −25 upstream of the UCP1 5′UTR),

Left primer:

(SEQ ID NO: 23) 5′-TCGTAAGCTTAGAGGCGGCGGCTGCAGACGGAGCGCGGTGTT-3′

Right primer:

(SEQ ID NO: 24) 5′-ACGAAGATCTCATTACCCCAAATAGCATCACA-3′

ii) the proximal targeted promoter/enhancer region (3685 bp upstream ofthe −25 nucleotide of the UCP1 5′UTR)

Left primer:

(SEQ ID NO: 25) 5′-TCGTAAGCTTAGAGGCGGCGGCTGCAGACGGAGCGCGGTGTT-3′

Right primer:

(SEQ ID NO: 26) 5′-ACGAACCGGTCAGAAGTGGTGAAGCCAGCCTGC-3′

iii) the distal targeted promoter/enhancer region (2435 bp upstream ofthe proximal targeted promoter/enhancer region) as indicated:

Left primer:

(SEQ ID NO: 27) 5′-TCGTACCGGTACAGGCTCTGGGAAGTAGGAGAAAGT-3′

Right primer:

(SEQ ID NO: 28) 5′-ACGAAGATCTCATTACCCCAAATAGCATCACA-3′

Each primer contains a restriction site to facilitate subsequent cloningin mammalian expression vector (see below).

Cloning of the promoter/enhancer in PCR reaction was performed with 500ng of BAC #RP11-5K16 as template, using Takara Ex Taq DNA Polymerase kit(Clontech) for amplification. PCR program steps were as follow:Initialization step, 92° C. for 2′, followed by 28 cycles: denaturation:92° C.-30 seconds/annealing: 59° C.-40 seconds/extension: 68° C.-5minutes 30 second, with a final elongation step 68° C.-8 minutes.

The full promoter/enhancer, proximal or distal promoter/enhancer weresubsequently subcloned in the reporter/MAR element-containing vectorp1_(—)68_GFP at the BlgII/HindIII sites, replacing the SV40 promotercassette [25]. Alternatively, the luciferase-based pGL3 Basic vector(Promega) was also used as another reporter type, using the sameBglII/NcoI sites for subcloning purpose.

The human UCP1 promoter sequence cloned was confirmed bystate-of-the-art sequencing, performed at biotechnology company,Fasteris SA, Switzerland. The sequence of the human UCP1 promotersequence is provided as follows (SEQ ID NO. 29):

5′-CATTACCCCAAATAGCATCACATTCTATCTCTGGATCACCATTTTTACACTTATCTAGAATTTGCCCACCTGTAGTTTCCACTCTTCGGCACTAATTATTTTGCTTAATTGCGTACAGAACAAATCTACCCCGTCCACTGTCTATGCCTTCAAGTATCTGAGAACAGTAATGTCCTGTTCGGTAAGTCATTTTCTCCTTTTCACTCTCTGGTCCTTCCATGGGGCTTCAATCCCCATACACCTCTTTTTTCTAAATTTCATAGGTCAGTTTTCCTGTCTCTTCTACCAGGTTCTACTGAAGATGAAAAAAAGTGCTTTTTTAAACCAAAAGTATTGCAATGTTTATTTTATCTTTGTAAGTTCCTTAGTAATATATACAAATCAAGTAAAAGATATATGTTGCATGTGATATTTTAACTTTTGATATGACTTATTGAAAAAATATATAAGGATACATAGCCATTGTGTGTCTTCAAATCATAGGAAAGTATCATGTCGCGAATGTATTGGGAAGGCAGTTGGGGTATCACGTAGTAGTTGAGAGTTAGGGGGTCAGGCAGATCCTCAGTGTACCATTTACTGGTTCCGTGACCTAGGAGAAGTTATTTAACTTCTCTGAGCTCTCTGAGTTTCCTCATCAGTGAAGGGGAATAACAATAATATATGCCTCCAAAGGCCGCAATGAGGACTAACTGTGTTAAGTTTTGTAAAATGCCTAAAATATTATAGTGTCTGGCACTTGTTCAATGCTATGTATTTGTTAAATACATGACATGAATAAATCTTTCATTGAGTTATGAGGATTAGGTACATCAGGTGCTTAGCATAAAGAGTGATTTATTAATAAGAATAGGCTCATGATGCAGGAATATTCATCACATATGTAAATAATCTGAAGCTCAGAGAAGTTAAGTAATTTGGCCATGCTTACCCAGTCAGTTATTATCTTAGTGAGAATTTGAACATGGGCCTCCTGGTCTCTTAATCACCATGCTATACCACTTATATCAGCATAGAAATGGAATATTTTCTCCTTAACGCAGAGTTTGATAGTCTTTGTCTCTTTGTATTGGGCTGGACTAAGAAAACCCAATCCTGTCCTCTTTCTACTTTTTCTCTGTTCCTAAGAGCACTCCCCTTTCTCTGTTGTATATCAGTTCCTAATGGTAGACACTTGAGCACCACTATTCTGTACAGCTCTCCGACAATCCCACATCTAGATGCCAAGCTGAGGTTGGCATTCTCACTAATTTGCTGTTATAAATATTAAGCTATCATAAGCGTTAGCCTACATATGACTCTTTCATATGTTAGTTAATTATTTTAGGGTAGAAATCCAAAAGTGGAGTTACCAGAAGTGGATATAGACATTCTGGCTGGGTGTGATGGTTCATGCCTGTAATCCCAGCACTTTGGGAGGCAGAGGCAGGCGGATCACTTGAGGCCAGGAGTTTGAGATCAGCCTGGGCCAACACAGCGAAACCCCATCTCTACTAAAAATTCCAAAACTAGCCAGGCATAGTGGCACATGCCTGTACTCCCAGCTACTTGGGAGGCTAAGACACAAGAATCGCTTGAACCCGGGAGGGAGGTGGAGGTTGCGGTGAGCTGAGATTGTGCCACCGTACTCCAGCCTGGGTGACACAGCTAGACTCTGTTTCAAAAAAAAAAAGAAAAAGAAAAGAAAAAAATAGACTTTCTCTTGGCTCAGTGTATACTGCCAAATTGTTTTCCAAAAAAATTGTGTCAATGTATAACACCATCACTAATATAGTATTGATATTATGGTTATTACATTTTAAAATTCATAATTTGTAATTATAACATTCATAATTTATTACTATTTATAATATTAATGTAAATGTATATTATATATAAATGTTATAGTAATTATAACTTTGGTAGTGACAAAGTATTAATTTATTAGGTGAAGTATATGCTTTTTTATTAGTGATAATAAATATATCCTCTCTCCCATTATAAAAGTTTGTATTTCTTCTTTTAGAAATTGATTCTTCTGTCATTTGCACATTTATCTGTATAATTATAACAGGGTATTTCCCAGTGGTGGCTAATGAGAGAATTATGGGAAAGTATAGAACACTATTCAAATGCAAAGCACTGTATGATTTTTATTTAATAGGAAGACATTTTGTGCAGCGATTTCTGATTGACCACAGTTTGATCAAGTGCATTTGTTAATGTGTTCTACATTTTCAAAAAGGAAAGGAGAATTTGTTACATTCAGAACTTGCTGCCACTCCTTTGCTACGTCATAAAGGGTCAGTTGCCCTTGCTCATACTGACCTATTCTTTACCTCTCTGCTTCTTCTTTGTGCCAGAAGAGTAGAAATCTGACCCTTTGGGGATACCACCCTCTCCCCTACTGCTCTCTCCAACCTGAGGCAAACTTTCTCCTACTTCCCAGAGCCTGTCAGAAGTGGTGAAGCCAGCCTGCTCCTTGGAATCCAGAACTACTTTCAGAATCTTGAACTTCTGTGACCTCTCAGGGTCCCCTTGTGTGAAGTTTTTGACGTCAGCTTCTCCTGTGACCCTTAGAAGTCACTCTTGTGTCTAGCACATCCCAGGTGCTCAGTCACCATTGAACTACAGTCATACTATCTCCTGGCAAAGGCTCTTAACTGTCCATGTTAGCCTGATATTAATATCCTGGAAGCTTATACTGTCGTTCTTCCTTCCAGGTTTAAATAAGGCAGCCCCTTTATCCTGTCACAGGTCCTCTCTCCCTACCTATCCTTACCTGTTTTGGATAACAACCTTTCTTCATTTCTAATAGATTTATTTATTTCTCACATTTCCTTCCCTTATCATAGTTTTCCTCTCACTTTCTCCTCTAGTTTGTCATACTCTGGCTTTAAAACATGCAAACATGTGCCTTATGGGGAAAAAAAGACAATTTTAATTTACCTTGCTTCTTTACAAATGTATTGTGGCTTCTTCTTATAGTCCAAATCTAAAACTCTTTACCCACCCACTGCCTTGAACTCCTTCCTCGTTGTGAAAGTAGGATGGGGCAAAGAGAGAATGCATGCCCCTCCCAACTGCTCAAACAAGTAAAGGTGCTGTTACAGTTATCTTTTGCTACCTTAATACAATAATTATTTTATTATATCTCACAATTTTATGGATCAGGAATTTAGACTGGGCTCAGCTAGGCGATTCTTCTGCTTTACTGACATCATAGGAGATCACTTGGTGGTATTCAACTGTCAGGTAGGCTTATCTGGAGGGTCCAAGATAGCTGTACTCTGGTGCCTGGTGCCTTGGTAAAGAGGGATGATGATGTGGGGCCTCTCCAGCATGAACAGCCTCAGAGAAGTTTGCTTTCTTACATGCTGGCCCAGGGCTCCAAGAGCAAATGTTGCAGTGAGTAAAGCAGAAGATACAAGGACTTTTATAATCTGGTCTCAGAAGCCACATGGCATCAGTTCTGTATTATTCTATTGGTCAAAACATTCATAAGCCTGCCAGATGCAAGGGGAAGGCATATGTACCCTCATCTTTTGATGGGAGGAATGTGATGGATTTGCAATTATGTTTTAAAACTACTACAGACAGAACCACTGAGAAAGATTCATGGGTAGCTTTGGGGTGAGGACTGGGAATTAACCTGTTGATAGCAGAGGTTCACTAGAGTCAACAAGGAATAAGGTCTCCTCTTGTACACTTTAGTCATACTATACCAACATTCTTAACCACTGCTTAGCCATCAGCCTCACAACATAACAACTCCATCATAGTTGTACTCCCTAAGATCACCAACAATGTTAGAGTCAAATCCGGTAGGTTTTTCTTTGTTTTTGTCCTCCTGACATTTTTTCTAAACTTGACACTGGTCAGACCCAATCTTTCTTTAATCATATTCTTAAATACCAGTTCTATCACTGGATATGTTACTGTTTCTTGTTCTCACTCTACCTTTGACAAAGCCATTCTTTCCAGACTATAACTCTGGGTCTGGGTCCCCCTATGGTTTGGCCCTTGAATTCTTTTCCTAGTCCTATTTGACTAGCCCCATTTTCCCGTGAAAAGCATGCCCCTTTCATTGCATCCATATCATGACTACCAAATACCTCCTCTATTTCTTCCTCTTTTAGCATGTTAAATGCAGCTTCCTAAGCTCTCTATCTGGATATCAACAGTATTCTCTCCAAATAATTCTAAGACTTTAAAAATTGGTTTAATCTTCTTACCCCTAAAATCACCCCCCTTACCAACTGCCTCATGACAATCATTGGTACTGTCACTGAGCTTGCAACCCATGTTCTTAAACATAGAGTAATCTTTGACTCCACATCTAATCATTCATAAAGCTGTATTGTCTATCAAATTAAATCTGACATTTATGTGAGAGCACTTCATAGTCTGTAAAGCACTACACAGGTGATAACATGAAGCTACACTCATAATGGATTTGCAGGCTCTGCTTCTCATTTGGCTTCTACAGCCTCATCCCTCACCAACTTCTTGCCCTACCTCTCTCTTTCTTCCCCATCACCCAATTTCCCAGTCAGTCAGGCCAACAGAATGCATTCTATATACGCGACTTGCTTTCCCCAACATCTTTGCCTGTATGCATGCCACTTATTTGCCTCAGTTGATCTTTATTTCAACAAGTGTTTGCAGAGGAGAAACCTCGCTGGCTCCTTCTCCTTTCTATTTTTTTTCAGAGGCTACCCGTCAGGTCAACATTGCCTTTTTCAGGGAAGCTCTGCAAGCCTGACCTCCCTTGGAAGTGCCTTAGGACTGGCTTCTTGCACAGTACACAACCTTTACTTATAGAGGGTTTGGAGATTATTCTTTATTCATGTCTTATTTCTCCTGCTCCTGGAGGAGATGACTCTGACTTCCACTGACTCTTTTGGGGGGCTTAAGTCAGGGTTGAGTACCAGAGGCCCTAAATAGCTGGACGTGGATTCTGGTAATATCAAATCCATCTTTGGCTTAACTGAGAGGTTCTGAAAGCTGGGACCTGACCTTGTCCATTTCCCTCTTTCTCCAGTTTCCTATTATTTCCCACTGTTTTTTTTAAAAGTTTTTTGTTTTCTTAAGTTTTCACAAGAATAAACATTGAAAATAAAATTTGCACAAAGATCGAACTAGGAAAGGCCACACAACCAACACATATTACATCATTATAGGTAAGTTAGCAGGGAGATTTCAGACCTGGGCTAGCTCTGGAACCACATTTTACACTGTTGAAAATAAAAGCTGGAGTACAGATGACTTTCCCAGGTTCACAGAGTTGGTAAGCTGGAGAGCTGCACCTGGAGCCAAGCAACCTGCCCTGTCCTTTCCACTGCACCCTCTAAGAAATCTAATTAGAAGGAACAGGTGGTATCTCATTTTGTACGGTGCTTTAGCAATGTACTATTTGCTTTCTAGTGTGTCTATTGTCTCGTTTGACATCTTCTCTCAAAAAGTGATGAAACGAAACGCTCTTTTTGACAAGTTCAGAGTGCTCTTGGTTCCTGTGTGGGATTCTTCCAAGTCTGAATTTGGTAGTGGGAAGAGAAGGAATCCGGAGGAAGGAGGATGAGAAGTTTAAAGGAGAGGAAAGGGAAGCAGAGAAGGCCGCAAGGTGCCTGCAAGATGTCTGGGGAGTTGGAGGAATGGAAGAGTGCCCCGCTCTTCCTTCTGGGAGAGCTCCAGCTAGGCAGAACCTTTCACCAAGGCTCTGATATCGTGCTGGTTTCCGAAAGCCCCAGCCGAAGGTGTGCAGCCAAAGGGTGACAGAAGGTGAGGCACGTGCGGGGGCGCGGGTGCTGACCGCCGCGGTGCGCCCTCCCTCCGACGTGCGGTGTGCGGGGCGCAGACAACCAGCGGCCGGCCCAGGGCTTTCGGGGAGCGAAGCAGGGCTCCCGAGGCACCGAGCGAGAATGGGAATGGGAGGGACCCGGTGCTCCCGGACACGCCCCCGGCAGGTCCCACGCCCGGGTCTTCTGAGACCTCGCGCGGCCCAGCCCGGGAGCGGCCCAGCTATATAAGTCCCAGCGGAAGACCGGAACGCAGAGGGTCCTGCTGGCGCGAGGGTGGGTAGGAGGGGACGCGGGGACTCGGCCCCCAACACCGCGCTCCGTCTGCAGCCGCCGCCTCT-3′

The section headings and subheadings used in this specification are fororganizational purposes only and are not to be construed as limiting thesubject matter described in any way. Further, while the presentteachings are described in conjunction with various embodiments, it isnot intended that the present teachings be limited to such embodiments.On the contrary, the present teachings encompass various alternatives,modifications, and equivalents as will be appreciated by those of skillin the art.

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The invention claimed is:
 1. A method for identifying an agent that induces differentiation of a brown adipocyte progenitor cell into a brown adipocyte, comprising: providing a brown adipocyte progenitor cell derived from skeletal muscle, wherein said brown adipocyte progenitor cell is positive for CD34 marker and negative for CD146 marker; contacting the brown adipocyte progenitor cell with an agent; determining in the brown adipocyte progenitor cell the presence of an indicator of differentiation into a brown adipocyte induced by said agent.
 2. The method of claim 1 wherein the indicator of differentiation is an increase in one or more of the following: expression of uncoupling protein-1 (UCP1) protein or mRNA, expression of mitochondrial transcription factor A (mtTFA) protein or mRNA, expression of peroxisome-proliferator-activated receptor-γ (PPARγ) coactivator-1α (PGC-1α) protein or mRNA, uncoupled respiration, glucose utilization rate, fatty acid oxidation rate, and a combination of any of the foregoing.
 3. The method of claim 1 wherein the brown adipocyte progenitor cell is capable of binding an antibody specific to said CD34 marker.
 4. The method of claim 1 wherein the brown adipocyte progenitor cell is further negative for CD45 marker.
 5. The method of claim 1 wherein the brown adipocyte progenitor cell is further negative for CD56 marker.
 6. The method of claim 1 wherein the agent is one or more of the following: a peroxisome-proliferator-activated receptor (PPAR)γ activator, modulator or inhibitor; a PPARα activator or modulator; a PPARδ activator or modulator; a dual PPARα and PPARδ activator or modulator; a pan-PPAR (α, δ, γ) activator or modulator; a phosphodiesterase (PDE)4 inhibitor; a PDE7 inhibitor; a nuclear receptor-interacting protein 1 (NRIP1) (RIP 140) inhibitor, a phosphatase and tensin homolog (PTEN) inhibitor; an α1-adrenergic full or partial agonist; a retinoid receptor α (RXRα) activator or modulator; a PPARγ coactivator (PGC)-1α activator; a PGC-1β inhibitor or activator; adiponectin or an activator of adiponectin receptor AdipoR1 and/or AdipoR2; a nitric oxide synthase (NOS) inhibitor or activator; a Rho kinase-ROCK inhibitor; brain-derived neurotrophic factor (BDNF); a monoamine oxidase (MAO) A inhibitor and/or an MAO B inhibitor; an activator of SRC proto-oncoprotein; an inhibitor of epidermal growth factor receptor (EGFR); an inhibitor of fatty acid amide hydrolase (FAAH); an inhibitor of mitogen-activated protein kinase (MAPK) 1 or 2 or 4 or 5 or 7 or 8; an inhibitor of cyclin-dependent kinase 9 (CDK9); a TGR5 agonist; an adenosine monophosphate activated protein kinase (AMPK) activator; bone morphogenetic protein 7 (BMP-7); an mammalian target of rapamycin (mTOR) inhibitor; an adenylate cyclase activator; or combinations of any of the foregoing.
 7. The method of claim 1 wherein the agent is a peroxisome-proliferator-activated receptor (PPAR)γ activator, modulator or inhibitor.
 8. The method of claim 1 wherein the agent is rosiglitazone.
 9. The method of claim 1 wherein the indicator of differentiation is an increase in one or both of uncoupling protein-1 (UCP1) protein and UCP1 mRNA.
 10. The method of claim 1, wherein the brown adipocyte progenitor cell is capable of expansion and passage in a culture.
 11. The method of claim 1, wherein the skeletal muscle is one or more of fetal, juvenile, and adult human skeletal muscle.
 12. A method for identifying an agent that induces differentiation of a brown adipocyte progenitor cell into a brown adipocyte, comprising: providing a plurality of brown adipocyte progenitor cells derived from skeletal muscle, wherein said plurality of brown adipocyte progenitor cells are positive for CD34 marker and negative for CD146 marker; contacting the plurality of brown adipocyte progenitor cells with an agent; determining in the plurality of brown adipocyte progenitor cells the presence of an indicator of differentiation into a brown adipocyte induced by said agent.
 13. The method of claim 12 wherein the indicator of differentiation is an increase in one or both of uncoupling protein-1 (UCP1) protein and UCP1 mRNA in the plurality of brown adipocyte progenitor cells.
 14. The method of claim 12 wherein the agent is one or more of the following: a PPARγ activator, modulator or inhibitor; a PPARα activator or modulator; a PPARδ activator or modulator; a dual PPARα and PPARδ activator or modulator; a pan-PPAR (α, δ, γ) activator or modulator; a PDE4 inhibitor; a PDE7 inhibitor; a NRIP1 (RIP 140) inhibitor, a PTEN inhibitor; an α1-adrenergic full or partial agonist; an RXRα activator or modulator; a PGC-1α activator; a PGC-1β inhibitor or activator; adiponectin or an activator of adiponectin receptor AdipoR1 and/or AdipoR2; an NOS inhibitor or activator; a Rho kinase-ROCK inhibitor; BDNF; a monoamine oxidase (MAO) A inhibitor and/or an MAO B inhibitor; an activator of SRC proto-oncoprotein; an inhibitor of EGFR; an inhibitor of FAAH; an inhibitor of MAPK 1 or 2 or 4 or 5 or 7 or 8; an inhibitor of CDK9; a TGR5 agonist; an AMPK activator; BMP-7; an mTOR inhibitor; an adenylate cyclase activator; or combinations of any of the foregoing.
 15. The method of claim 12 wherein the agent is rosiglitazone.
 16. The method of claim 12 wherein said plurality of brown adipocyte progenitor cells are capable of binding an antibody specific to said CD34 marker.
 17. The method of claim 12 wherein said plurality of brown adipocyte progenitor cells are further negative for CD45 marker.
 18. The method of claim 12 wherein said plurality of brown adipocyte progenitor cells are further negative for CD56 marker. 